The long-term objective of the research described in this grant proposal is to better understand metabolism-based drug-drug interactions that are underpredicted from in vitro data. This is important because underprediction of effect has negative consequences for drug therapy and the development of new drugs. It is also important because these outliers represent a significant fraction of clinically relevant drug-drug interactions. An in-depth understanding of why predictions fail should improve predictive efforts. The interactions we propose to study are drawn from the larger class of interactions that appear to arise as the result concentrative cell uptake and/or metabolites of the interactant drug. Predictive quality is degraded because the magnitude of the inhibition depends not only on the concentration of the interactant drug at the active site, but also on the activity of the enzymes involved in converting the drug to the proximate inhibitory metabolite as well as the mechanism of metabolite inhibition. The first section of this proposal investigates two potent inhibitors of P450-catalyzed drug metabolism, fluvoxamine and itraconazole, who's in vivo effects are 10- to 100- fold higher than predicted. Fluvoxamine causes potent and differential inhibition of at least 4 important human P450' s in vivo. Aims 1-3 will identify the sources of the underprediction for each enzyme by examining the importance of concentrative uptake as well as direct inhibitory effects of fluvoxamine, itraconazole and their major metabolites on enzyme activities in microsomal preparations and human hepatocytes. The finding that time dependent, persistent inhibition of CYP3A4 activity by itraconazole is observed in microsomes and intestinal cells further suggests that itraconazole is a mechanism-based inhibitor of this important enzyme. Aim 4 will assess if other factors, such as concentrative uptake, are required to fully explain the effect of itraconazole and, if so, to identify them. The second section of the proposal addresses the propensity of alkylamine containing drugs to elicit irreversible inhibition of enzyme activity via formation of MI complexes in vivo and in vitro. This process requires that the alkylamine undergo as many as 4 rounds of oxidation to the corresponding C-nitroso metabolite prior to the inhibitory event. This very complex type of inhibition has not been fully characterized and a deeper understanding of the underlying mechanisms is critical for meaningful and reproducible in vitro-in vivo predictions of inhibitory effect. Aims 5 and 6 of this proposal set out to fully describe the kinetics of the system and evaluate the limits and advantages of a less complex empirical approach.